Six conserved amino acids are found in an alignment of E. coli RNase H with the C-terminal region of Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) and other retroviral pol sequences. Functional significance of selected amino acids in the putative RNase H domain of reverse transcriptase (RT) was determined in partially purified cloned M-MuLV RT expressed in E. coli and in infectious molecularly cloned M-MuLV after amino acid substitutions were made by site-directed mutagenesis. Modification of either of two conserved aspartic acid residues resulted in greater than 25-fold loss of RNase H activity. One of these mutant enzymes exhibited normal RNA dependent polymerase activity, whereas polymerase activity (dTTP and dGTP in corporation) was reduced 50% in the other. MuLV proviruses containing either of these modifications were replication defective. Infectious ecotropic virus phenotypically similar to M-MuLV could be rescued by co- infection or co-transfection with competent amphotropic or MCF MuLVs. These findings support the localization of the RNase H domain in RT, show that a single amino acid substitution can selectively suppress RNase H activity, and confirm the requirement for RNase H for viral replication. Genetic recombination between two defective Human Immunodeficiency Virus (HIV) sequences has been demonstrated. Three different replication defective proviruses constructed by site-directed mutagenesis of the tat, int, or env regions of an infectious HIV molecular clone were used. A cell line, 8E5, carrying a pol defective endogenous HIV provirus was transfected with each of three mutants. Infectious virus was recovered after transfection with tat or env mutants and were plaque purified. Restriction mapping of viral DNA isolated from cells infected with these biologically cloned recombinant viruses indicated multiple crossing over events had occurred to generate infectious virus.